Automatic Liquid Transfer Optimization Pipetting Apparatus and Method

ABSTRACT

An automatic liquid transfer optimization pipetting apparatus and method is disclosed. Namely, a liquid handling apparatus includes a pump supplying a nozzle (i.e., a pipette tip) via a conduit, one or more pressure sensors, and an electronic controller, and wherein the pipette tip is submerged in a liquid. Further, a method of automatic liquid transfer optimization pipetting includes the steps of actuating the pump to move a designated volume of liquid and then allowing the system to settle to a steady state after completion of pump actuation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to and claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent App. No. 62/611,261, entitled“Automatic Liquid Class Pipetting Apparatus and Method,” filed on Dec.28, 2017; the entire disclosure of which is incorporated herein byreference.

TECHNICAL FIELD

The presently disclosed subject matter relates generally to liquidhandling methods and more particularly to an automatic liquid transferoptimization pipetting apparatus and method.

BACKGROUND

Automated liquid handling instruments include robots used to transferspecific quantities of liquids, such as reagents or samples, betweendesignated containers. Liquid handling instruments are useful in avariety of applications including cell biology, genomics, forensics, anddrug research. The instruments assist humans with the repetitive task oftransferring liquids in a wide range of volumes to improve speed andefficiency of the operations, as well as precision and accuracy of thedelivered volumes. Examples of commercially available liquid handlinginstruments include, but are not limited to, the Freedom EVO series byTecan Trading AG (Mannedorf. Switzerland), the Microlab VANTAGE Systemby Hamilton Company (Reno, NV), and the JANUS Workstation series byPerkin Elmer (Waltham, MA).

The advantages of automating liquid handling processes includeincreasing throughput and efficiency of operations and eliminating humanerrors. The advantages are contingent on the accuracy and repeatabilityof pipetting operations to ensure the integrity of experimental resultsin the above mentioned fields.

To achieve good performance, the control parameters of a liquid handlinginstrument must be tuned for particular types of liquids. Controlparameters can be numerous, examples include the rate of pump actuation,volume to be actuated by the pump, depth of probe immersion in theliquid, delay of probe removal from the liquid, and speed of proberemoval from the liquid.

A liquid handling instrument can have a default set of controlparameters, which is useful for pipetting water or aqueous solutionsthat have very similar properties to water. However, if the instrumentis to be used with a liquid that has substantially different propertiescompared with aqueous solutions, the control parameters must becalibrated and tuned to ensure accurate and precise pipetting of such aliquid. Calibration of new control parameters is a complex and timeconsuming process and is typically performed by a specialist. Moreover,the user of the instrument is required to explicitly specify the set ofcontrol parameters associated with the liquid to be pipetted at the timean automated pipetting protocol is authored. If the control parametersare selected incorrectly, the accuracy and precision of the resultingpipetting operation can fail to meet the required specification.

These requirements stand as a barrier to the use of advanced liquidhandling instruments for the average lab worker. Namely, calibration ofnew control parameters for a particular liquid requires time and specialtraining. Further, programming of liquid handling operations requirestraining and experience. Thus, there is a need to simplify the userexperience of automated liquid handling instruments with respect to thedetermination and selection of control parameters.

Relevant patents to this background include the following:

U.S. Pat. No. 8,357,544, entitled “Method for selecting pipettingparameters for a liquid.” The ’544 patent describes a liquid-basedliquid handler wherein the liquid handler can measure pressurethroughout an aspiration and create a pressure curve from themeasurements. Other aspects of the ’544 patent include comparing themeasured pressure curve to known pressure curves, and determiningdispense pipetting parameters (e.g., plunger speed, etc.) based on thecomparison of the measured pressure curve to known pressure curves.

U.S. Pat. No. 7,694,591, entitled “Method and apparatus for evaluating adosing operation.” The ‘591 patent describes a syringe and air-basedliquid handler that can monitor pressure throughoutaspiration/dispensing, detect errors if the signal differs from a knownand expected values, and identify certain errors.

U.S. Pat. No. 6,938,504, entitled “Method and device for evaluating aliquid dosing process.” The ‘504 patent describes an example ofmonitoring pressure throughout the aspiration or dispensation, andcomparing it to a predetermined set point range for that liquid and setof control parameters in order to evaluate for errors.

U.S. Pat. No. 6,662,122, entitled “Method for the controlledproportioning of liquids while dislocating a gas cushion.” The ‘122patent describes an example of monitoring pressure and time throughoutan aspiration and dispense, and using the known volume of gas within thepipetting system along with parameters of pump actuation to determinethe time of a pipetting operation and compare it to an expected time.

U.S. Pat. No. 7,197,948, entitled “Method of dosing liquid volumes andapparatus for the execution of the method.” The ‘948 patent describes aprocess that includes actuation of the pump during aspiration andwherein dispense is controlled to apply and maintain a particularworking pressure. The flow of liquid into the pipette tip is monitoredby calculations based on pressure measurements throughout this process.When the desired volume is achieved, the pump is actuated to abate thisworking pressure. Dispense parameters are determined based on theresults of the aspiration.

U.S. Pat. No. 8,096,197, entitled “Air displacement liquid deliverysystem and related method.” The ‘197 patent describes a process ofmonitoring pressure inside and outside the pipette tip to improve thespeed of viscous pipetting by moving the plunger beyond the set point tomaintain a greater flow rate, then moving it back to the set point.Also, the ‘197 patent describes monitoring the volume of liquid in thetip over time based on pressures.

U.S. Pat. No. 7,634,367, entitled “Estimating fluidic properties andusing them to improve the precision/accuracy of metered fluids and toimprove the sensitivity/specificity in detecting failure modes.” The 367patent describes a process of monitoring pressure during aspiration andextracting features from the sensed data to estimate viscosity. The ‘367patent is based on determined viscosity, and adjusting controlparameters to improve accuracy/precision. Calibration curves are createdwith different fluids of known viscosity. Unknown fluid viscosity can befound by fitting the pressure curve/features of the pressure curve tothose of known viscosities. The amount of volume needed relative toaspiration and extracting steps can be corrected based on the detectedviscosity.

U.S. Pat. No. 8,307,697, entitled “Method for estimating viscosity.” The‘697 patent describes a specific method of estimating viscosity duringaspiration. The method involves comparing the pressure change from justbefore aspiration (ambient) and immediately after plunger movement topressure change between immediately after plunger movement and somefixed time after plunger movement. Specifically, the ‘697 patentmeasures the rate of pressure change during plunger movement, and therate of pressure change after plunger movement. The ‘697 patent furthermeasures pressure a fixed amount of time after the plunger stops moving.Based on the measurements and calculations, viscosity is estimated basedon a calibration of the system. Next, an equation is calibrated todetermine the actual viscosity based on these pressure measurements.

SUMMARY

In an aspect, an apparatus for automatic liquid transfer optimizationpipetting is disclosed. In some embodiments an apparatus for automaticliquid transfer optimization pipetting comprises a pump; a pipette tipin fluid communication with the pump, the pipette tip comprising aconduit with a working air pressure relative to ambient pressure, theworking air pressure having an upper threshold; a pressure sensorconnected to the conduit, the pressure sensor being adapted to measurethe working air pressure, the ambient pressure, and changes to theworking air pressure caused by aspiration or dispensation of a liquid bythe pipette tip; and a controller in electrical communication with thepump and the pressure sensor, wherein the controller receives input fromthe pressure sensor, commands a velocity of the pump, and maintains theworking air pressure at or below the upper threshold by adjustment ofthe velocity of the pump during aspiration or dispensation of a liquidby the pipette tip.

In some embodiments, an apparatus described herein further comprises aframe; and an actuator fixed to the frame and in electricalcommunication with the controller, the actuator being operativelyconnected to the pump and the pipette tip and adapted to controlmovement of the pump, the pipette tip, or both.

A pump described herein can in some cases comprise a motor; and asyringe operatively connected to the motor.

In another aspect, a method of automatic liquid transfer optimizationpipetting is disclosed herein. In some embodiments a method of automaticliquid transfer optimization pipetting comprises providing an apparatusfor automatic liquid transfer optimization pipetting described herein;aspirating or dispensing a liquid in the conduit of the pipette tip;adjusting a velocity of the pump to limit the working pressure in theconduit of the pipette tip to a pressure level equal to or less than amaximum working pressure value during aspiration or dispensing of theliquid; actuating pump to displace a volume of air in the conduit, thevolume of air corresponding to a desired volume of the liquid beingaspirated or dispensed. In some embodiments, a method described hereinfurther comprises providing a container holding the liquid, the liquidhaving unknown physical properties; and inserting the pipette tip intothe liquid for aspiration or dispensation. In some instances, a methoddescribed herein can further comprise determining a reference pressurein the conduit, the reference pressure being a pressure in the conduitduring an absence of any liquid in the conduit. In yet otherembodiments, a method described herein can further comprise monitoringpressure within the conduit with the pressure sensor. A referencepressure in some cases is equal to an ambient pressure within theconduit and outside the conduit. In some instances, aspirating ordispensing a liquid comprises actuating the pump.

A method described herein can further comprise the step of filtering themeasurements by the pressure sensor by calculating a moving average ofthe pressure sensor signals. A moving average described herein can bedetermined by calculating the average of all prior measurements within aperiod of time.

A controller described herein can adjust the velocity of the pump duringactuation in some instances to maintain the working pressure in theconduit at a pressure level equal to or less that the maximum workingpressure.

In some embodiments, a method described herein can further compriseconfirming a steady state pressure response within the conduit after thepump has been actuated to displace the volume of air in the conduit. Insome instances, confirming the steady state pressure response comprisesmeasuring the slope of the pressure response over time. In some cases,the slope is calculated by dividing the difference in pressure beforeand after a time interval by the duration of the time interval, usingthe formula:

$s\mspace{6mu} = \mspace{6mu}\frac{P_{n} - P_{n - 1}}{t_{s}}$

wherein s is the slope (time rate of change) of the pressure response, nis the number of time intervals that have passed since the beginning ofslope measurement, P is the pressure, and t_(s) is the slope interval oftime.

In some embodiments, a pump described herein can comprise a syringe anda piston, and wherein actuating the pump comprises moving the pistonoutward from the syringe during an aspiration, and moving the pistoninto the syringe during a dispensation. The actuating step can be doneat a sufficient speed in some cases to create a substantiallymeasureable pressure change within the conduit.

A working pressure described herein, can, after a period of exponentialdecay of one time constant be calculated as:

P_(τ) = P₁-0.4 * (P₁ - P₀)

wherein P_(τ) is the working pressure, P₀ is the reference pressure, andP₁ is the pressure at the end of the piston movement and the beginningof the exponential decay. In some embodiments, a method described hereincan comprise the step of continuously monitoring the working pressure asthe working pressure decays until the value of the P_(τ) is reached.

In some embodiments, termination of a pump actuation is with adeceleration of the velocity of the pump that acts as a step input tocreate a substantially measurable first-order response to the workingpressure.

In some embodiments, a method described herein can further comprise thestep of determining parameters to adapt the liquid to the pipettingapparatus. The parameters can be calculated by:

t_(f) = c₁ * τ + c₂

t_(s) = c₃ * τ + c₄

s₀ = c₅ * τ + c₆

wherein t_(f) is filtering moving average time window, t_(s) is theslope determination time window, so is the slope threshold for steadystate, and c₁, c₂, c₃, c₄, c₅, and c₆ are empirically determinedcoefficients specific to a particular pipetting system design.

BRIEF DESCRIPTION OF DRAWINGS

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying Drawings, whichare not necessarily drawn to scale, and wherein:

FIG. 1 illustrates a block diagram of an example of a liquid handlingapparatus that can be used to perform the presently disclosed automaticliquid transfer optimization pipetting method;

FIG. 2 illustrates a side view of a specific example of oneinstantiation of the liquid handling apparatus of FIG. 1 that can beused to perform the presently disclosed automatic liquid transferoptimization pipetting method;

FIG. 3A and FIG. 3B show plots of an example of a measured signal andsystem response during aspiration of an aqueous solution using thepresently disclosed automatic liquid transfer optimization pipettingmethod;

FIG. 4 shows a plot of an example of a measured signal and systemresponse during aspiration of 100% glycerol using the presentlydisclosed automatic liquid transfer optimization pipetting method;

FIG. 5 illustrates a flow diagram of an example of a general method ofautomatic liquid transfer optimization pipetting using the presentlydisclosed liquid handling apparatus; and

FIG. 6 illustrates a flow diagram of an example of a specific method ofautomatic liquid transfer optimization pipetting using the presentlydisclosed liquid handling apparatus.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Drawings, in which some,but not all embodiments of the presently disclosed subject matter areshown. Like numbers refer to like elements throughout. The presentlydisclosed subject matter can be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein;rather, these embodiments are provided so that this disclosure willsatisfy applicable legal requirements. Indeed, many modifications andother embodiments of the presently disclosed subject matter set forthherein will come to mind to one skilled in the art to which thepresently disclosed subject matter pertains having the benefit of theteachings presented in the foregoing descriptions and the associatedDrawings. Therefore, it is to be understood that the presently disclosedsubject matter is not to be limited to the specific embodimentsdisclosed and that modifications and other embodiments are intended tobe included within the scope of the appended claims.

In some embodiments, the presently disclosed subject matter provides anautomatic liquid transfer optimization pipetting apparatus and method.The presently disclosed automatic liquid transfer optimization apparatusand method is capable of transferring liquids regardless of liquidproperties and with no prior knowledge of such liquid properties. Thepresently disclosed automatic liquid transfer optimization apparatus andmethod relates to automating certain aspects of liquid transferoperations on automated liquid handling instruments to simplify the userexperience of such instruments. As compared with conventional automatedliquid handling instruments, the presently disclosed automatic liquidtransfer optimization apparatus and method provides a means to reduce orentirely eliminate (1) the need to specify liquid properties whenprogramming an automated liquid handler, and (2) the need to calibratepipetting parameters for each specific liquid to be processed.

In some embodiments, the presently disclosed automatic liquid transferoptimization pipetting apparatus and method can be used to improve theuser experience of automated liquid handling instruments by automatingsome aspects of pipetting operations that can otherwise be explicitlyprogrammed by the user. For example, the automatic liquid transferoptimization pipetting apparatus and method can be used to bypassmeticulous calibration of the pipetting parameters related to variousliquid properties such that an automated liquid handling instrument canaccurately and precisely carry out a pipetting operation with no priorknowledge of the properties of the liquid to be pipetted.

In the current state of the art, the control parameters of an automatedliquid handling instrument must be specifically calibrated for eachpipetting operation, depending on the properties of the liquids to betransferred. This is a delicate and time consuming process that musttypically be performed by a trained specialist. In addition, after thecontrol parameters for a particular liquid have been calibrated, theuser is required to specify the types of liquids used in each step of apipetting protocol when programming an automated liquid handler. Suchinconvenience and complexity could deter the average lab worker fromusing an automated liquid handler for their pipetting tasks which arenormally carried out by hand. Accordingly, in some embodiments, thepresently disclosed automatic liquid transfer optimization pipettingapparatus and method can be used to reduce or entirely eliminate theseinconveniences and achieve accurate and repeatable pipetting with noprior knowledge of liquid properties. Namely, the automatic liquidtransfer optimization pipetting apparatus and method enables automaticreal-time adaptation of control parameters to suit whatever liquid thesystem encounters.

FIG. 1 is a block diagram of an example of a liquid handling apparatus 1that can be used to perform the presently disclosed automatic liquidtransfer optimization pipetting method. The liquid handling apparatus 1is a pipetting device and is an example of the presently disclosedautomatic liquid transfer optimization pipetting apparatus.

The liquid handling apparatus 1 includes a pump 2 in fluidiccommunication with a nozzle 3 via a conduit 4. The pump 2 is capable ofmetering specific volumes and can vary in mechanism. In one example, thepump 2 is a syringe pump that includes a syringe 22 that is driven by amotor 21. In one example, the pump 2 can be an air pump producing aregulated source of positive and/or negative (i.e., vacuum) pressure,which along with a flow sensor (not shown) and a valve (not shown) canbe used to meter volumes. In one example, the conduit 4 is filled withair. In another example, the conduit 4 can be filled entirely orpartially with a system liquid, such as water, which reduces the volumeof air between the nozzle 3 and the pump 2.

The nozzle 3 has an orifice 32 through which liquids are taken up intoor ejected out of the nozzle 3. In one example, the nozzle 3 can bepermanently fixed to the liquid handling apparatus 1. In anotherexample, the nozzle 3 can be a removable nozzle, such as a removable anddisposable pipette tip. Accordingly, the nozzle 3 is hereafter referredto as a pipette tip 3. In one example, the liquid handling apparatus 1includes one or more pressure sensors 9 in the conduit 4. The pressuresensors 9 are used to measure the air and/or liquid pressure in theconduit 4 and/or the pipette tip 3. The liquid handling apparatus 1further includes an electronic controller 7 that can further include anoptimization algorithm 8. The electronic controller 7 and/or theoptimization algorithm 8 can be used to process information from thepressure sensors 9 and to control actuation of the pump 2 (e.g., thesyringe pump).

Further, a container 6 is provided in relation to the pipette tip 3 ofthe liquid handling apparatus 1. The container 6 holds a quantity ofliquid 61. In the liquid handling apparatus 1, the orifice 32 of thepipette tip 3 is inserted into the liquid 61 so that it can aspirate ordispense the liquid 61.

A collection of control parameters exist that govern the execution of apipetting operation (e.g. an aspiration or a dispensation) by anautomated liquid handling device, such as the liquid handling apparatus1. Such control parameters can include the rate of pump actuation, thedelay between completion of pump actuation and removal of the pipettetip 3 from the liquid 61, and the rate of pipette tip 3 removal from theliquid 61. The control parameters must be modulated and optimized tosuit the properties of a particular liquid, in order to ensurerepeatable and error-free pipetting of that liquid. Accordingly, in someembodiments, the presently disclosed liquid handling apparatus 1 andmethod provide analysis of sensor data to adjust the pipetting controlparameters in real time in order to maximize pipetting performance withno prior knowledge of the properties of the liquid 61 being pipetted.More specifically, the liquid handling apparatus 1 and method recognizethe first-order system response of the air pressure in a pipette tip(e.g., pipette tip 3) during a liquid handling operation in order toensure error-free aspiration and dispensation, and to predict the timeat which the operation will be complete.

The presently disclosed liquid handling apparatus 1 and method can allowdetection of certain errors that would indicate a failed pipettingoperation. In a laboratory setting, a user with no experienceprogramming a liquid handler for use with particular liquids can easilyand intuitively, with minimal training, work with an automated liquidhandler on which the algorithms discussed herein have been implemented.Namely, a user with no experience programming a liquid handler for usewith particular liquids can easily and intuitively, with minimaltraining, work with the presently disclosed liquid handling apparatus 1and method in which the algorithms discussed herein (e.g., optimizationalgorithm 8) have been implemented.

Referring now to FIG. 2 is a side view of a specific example of oneinstantiation of the liquid handling apparatus 1 shown in FIG. 1 thatcan be used to perform the presently disclosed automatic liquid transferoptimization pipetting method. As illustrated, the liquid handlingapparatus 1 includes, but is not limited to, the pump 2, the pipette tip3 as described above (in FIG. 1 ) in airtight connection to the conduit4 leading to the pump 2, the electronic controller 7, the optimizationalgorithm 8, the pressure sensors 9, and a vertical linear actuator 5which can move the pump 2 and pipette assembly vertically.

In particular, FIG. 2 shows more details of one example of the pump 2.Namely, in this example, the pump 2 can be a syringe pump that includes,but is not limited to, the motor 21 that drives a lead screw 24, alinear motion guide 23, and the syringe 22. The vertical linear actuator5 can include, but is not limited to, an actuator motor 51 that drivesan actuator lead screw 53, an actuator linear motion guide 52, and allattached to a fixed frame 54 of the liquid handling apparatus 1.

The liquid handling apparatus 1 of FIG. 2 can be connected to anelectronic controller 7 in a manner as illustrated in FIG. 1 . Anelectronic controller 7 can, for example, be a general purpose computer,special purpose computer, personal computer, microprocessor, or otherprogrammable data processing apparatus. The electronic controller 7serves to provide processing capabilities, such as storing,interpreting, and/or executing software instructions, as well ascontrolling the overall operations of the liquid handling apparatus 1.The electronic controller 7 and/or the optimization algorithm 8 can becapable of, but not limited to, generating signals, receiving signals,processing signals, sending motion commands, and/or processing anyinformation or data in order to perform the electronic functionsdescribed herein as well as any other functions.

An air-displacement pipetting device, such as the liquid handlingapparatus 1 of FIG. 1 and FIG. 2 , can be theoretically modeled as asecond-order system including a mass, a capacitance, and a resistance. Asecond-order system includes two energy storage elements, usually in theform of a spring as the capacitance and the inertia of the mass, and canbe modeled with a second-order differential equation. The response of asecond-order system depends greatly on the nature of the components inthe system, but is an understood phenomenon. For example, a second-orderelectrical system can include an inductor, a capacitor, and a resistor.In such a system, energy can be stored in the capacitor and in theinductor. In a pipetting device, “mass” refers to the liquid volumewithin the pipette tip, “capacitance” refers to the compressible airvolume between the liquid in the pipette tip and the pump, and“resistance” refers to the restricted flow of fluid through the orificeof the pipette tip. Such a device can store energy in two modes: (1) inthe compressible air volume capacitance, and (2) in the inertia of theliquid.

FIG. 3A, FIG. 3B, and FIG. 4 are plots of examples of a measuredpressure response during aspiration of a liquid using the presentlydisclosed automatic liquid transfer optimization pipetting apparatus(e.g., the liquid handling apparatus 1) and method. More specifically,FIG. 3A shows a plot 300 and FIG. 3B shows a plot 310 of a measuredpressure response during aspiration of water, and FIG. 4 shows a plot400 of a measured pressure response during aspiration of 100% glycerol.

Referring now to the plot 300 of FIG. 3A and plot 400 of FIG. 4 , someaspects of the presently disclosed automatic liquid transferoptimization pipetting apparatus (e.g., the liquid handling apparatus 1)and method are explained. Plot 310 of FIG. 3B, will be explained in moredetail further below. In FIG. 3A, plot 300 shows a pressure response 800of the enclosed volume of air during device actuation. After actuationof the syringe pump 2 for the desired volume is completed, the pressureresponse 800 demonstrates an exponential decay 801 as it returns to thesteady state condition 802. Notably, the pressure response 800 settlesto the steady state condition 802 with no significant oscillation aboutthe steady state. This result shows that the liquid handling apparatus 1is an over-damped second-order system, suggesting the effect of the massinertia (e.g., momentum of the fluid entering or leaving the pipette tip3) is negligible compared to the effects of the resistance andcapacitance. Therefore, the liquid handling apparatus 1 can be modeledmore simply as a first-order system including a capacitance and aresistance in the form of the compressible air volume between the liquid61 in the pipette tip 3 and the restricted flow of fluid through theorifice 32 of the pipette tip 3, respectively. Actuation of the pump 2can be viewed as input to the first-order system.

For the purpose of describing methods disclosed herein, an aspiration ordispensation of liquid carried out by such a device (i.e., the liquidhandling apparatus 1 of FIG. 1 and FIG. 2 ) can be divided into twophases. During the first phase, the pump 2 is actuated to displace avolume of air in the pipette tip 3, which corresponds to a desiredvolume of liquid 61 that will move into or out of the pipette tip 3. Dueto the compressibility of the air in the conduit 4, the movement ofliquid into or out of the pipette tip 3 will lag the actuation of thepump 2. Therefore, at the time pump 2 completes its actuation, the airin the conduit 4 will exist in a compressed or expanded state, havingstored energy in the form of pressure or vacuum relative to the ambientpressure outside the instrument, thus causing liquid to continue movinginto or out of the pipette tip 3. The second phase begins at this time,upon completion of the pump actuation of the desired volume, andcontinues until the air pressure in the conduit 4 settles to equilibriumwith the air pressure outside the instrument, at which time liquid is nolonger moving into or out of the pipette tip 3. At this time theaspiration or dispensation is complete and the pipette tip 3 can beremoved from the liquid 61.

The pressure or vacuum of the enclosed air volume in the device relativeto the ambient pressure outside the system during a pipetting operationwill be referred to herein as the “working pressure.” In the specificcase of vacuum inside the system relative to outside the system, asduring an aspiration, the working pressure will be specified as“negative working pressure.” In the specific case of elevated pressurein the system relative to outside the system, as during dispensation,the working pressure will be specified as “positive working pressure.”

During the first phase of a pipetting operation, the pump 2 is actuatedand a change in working pressure grows in the air volume within conduit4. If the working pressure is allowed to grow too much, errors can occurresulting in inaccurate pipetted volumes. During aspiration it ispossible for the negative working pressure to become so severe that thepressure inside the pipette tip 3 drops below the vapor pressure of theliquid 61 being aspirated, causing the liquid 61 to vaporize and producegas bubbles in the pipette tip 3. This type of error can be referred toas “cavitation,” and results in poor accuracy of transferred volumes.During a dispensation, the liquid 61 inside the pipette tip 3 should beallowed to flow smoothly and cohesively out of the pipette tip 3. If thepositive working pressure is too severe, the liquid 61 in the center ofthe pipette tip 3 can flow faster than the liquid coating the insidewalls of the pipette tip 3, causing the pressurized air within theliquid handling apparatus 1 to escape the pipette tip 3 rather than thefull desired volume of liquid. This type of error can be referred to asto as “tunneling,” which also results in poor accuracy of transferredvolumes.

During the first phase of a pipetting operation, some methods disclosedherein relate to limiting the energy stored in the capacitance bylimiting the working pressure in the conduit 4, as measured by thepressure sensor 9 during pump actuation, so as not to cause pipettingerrors. By limiting the working pressure of the air volume withinconduit 4 during the first phase of a pipetting operation, cavitation ortunneling can be avoided regardless of the properties of the liquid 61being aspirated or dispensed. The working pressure can be limited byadjusting the rate of pump actuation during the first phase of apipetting operation.

During the second phase of a pipetting operation, some methods disclosedherein relate to predicting and confirming the correct time at which theworking pressure settles to equilibrium with ambient pressure outsidethe liquid handling apparatus 1, which means that liquid is no longerleaving or entering the pipette tip 3 and the pipetting operation iscomplete. If the pipette tip 3 is removed from the liquid 61 before theworking pressure settles to steady state, pipetting volume errors canoccur because the total volume of liquid corresponding to the volumeactuated by the pump has not been fully aspirated or dispensed from thepipette tip 3. If the pipette tip 3 remains in the liquid 61 for anytime after equilibrium has been reached, it reduces the efficiency ofthe operation. Thus, methods described herein comprise confirming in atimely manner that an equilibrium state has be achieved, so that theexecution of the pipetting operation can proceed efficiently.

In some embodiments, a method of limiting the working pressure duringthe first phase of a pipetting operation is described as follows. In oneexample and referring now again to plot 300 of FIG. 3A and plot of FIG.4 , a method described herein begins with measurement of a referencepressure 803 (P₀) prior to an aspiration or dispensation. For example, areference pressure 804 can be a pressure in the conduit 4 when there isan absence of liquid in the conduit 4. The reference pressure 803 ismeasured at a time before the syringe pump 2 is actuated, but can bemeasured before or after the pipette tip 3 is lowered into the liquid61. In one embodiment, the reference pressure 803 can be calculated asan average of numerous measurements in order to filter the effects ofnoise on the signal.

Methods described herein proceed with actuation of the pump 2 todisplace a volume of air in the pipette tip 3 associated with thedesired volume of liquid to be aspirated or dispensed. The initialcontrol parameters of pump actuation, such as pump rate, are the samefor any pipetting operation, regardless of the liquid 61 to betransferred. Preferably, the default control parameters for pumpactuation provide a sufficient pump rate to create a substantiallymeasurable change in working pressure during any pipetting operation. Inone embodiment, the initial control parameters can be determined basedon the volume of liquid to be aspirated or dispensed.

During pump actuation the working pressure within the liquid handlingapparatus 1 is monitored. Liquids of lower viscosity will normally haveless resistance to flow through the orifice 32 of the pipette tip 3, sothe working pressure cannot increase considerably during pump actuationwhen pipetting such liquids. Liquids of higher viscosity will tend tohave more resistance to flow through the opening or orifice 32 of thepipette tip 3, and thus the working pressure can increase to levels thatwould cause pipetting errors during pump actuation. If the workingpressure approaches a predetermined threshold 804 (see plot 400) duringpump actuation, the movement of the pump 2 is slowed or stopped toprevent the working pressure from growing to a point at which the errorsdescribed above can occur. The actuation of the pump 2 is controlled sothat the working pressure does not exceed a predetermined threshold.

In some embodiments, the motion of the pump 2 can be halted entirelywhen the working pressure reaches the threshold. At a time the pump 2 ishalted, e.g., at pump halt time 805 (see plot 400), the working pressurewill immediately begin to decay as liquid enters or leaves the pipettetip 3. Actuation of the pump 2 can begin again when the working pressuredecays beyond a lower threshold 806 (see plot 400). In some instances,actuation of the pump 2 can be paused and resumed multiple times inorder to complete the full actuation of the pump 2 required to deliverthe desired volume without exceeding the working pressure upperthreshold. The values of the upper and lower thresholds are related tothe design of a particular pipetting device and pipette tip 3 and can beempirically determined.

In one example, the velocity of the pump 2 is adjusted by a controlloop, which considers working pressure as an input and pump velocity asan output. In this example, if the working pressure approaches the upperthreshold during pump actuation, the velocity of the pump 2 is adjustedby the control loop so as to maintain the working pressure at or belowthe upper threshold. Such a control mode can allow for faster executionof a pipetting operation.

The pump 2 is halted when it has displaced the volume of air required toaspirate or dispense an associated volume of liquid. It is important tonote that the pump 2 should never be actuated to displace more volumethan that associated with the desired volume of liquid to be aspiratedor dispensed.

The second phase of a pipetting operation begins at a time when the pump2 completes its actuation for the desired volume, e.g., at a pumpactuation completion time 807. The halting of the pump actuation is achange of input to the first-order system, adjusting input from somepositive or negative value to zero. If the pump motion is halted withsufficient deceleration, the change in input can be estimated as a stepinput. The response of a first-order system to a step input ischaracterized by the time constant parameter, a principle understood inthe study of signals and energy system models. The time constant is aperiod of time that describes the speed with which a system responds toa change of input. The time constant is typically considered as theperiod of time required for a system to reach 63.2% (equal to 1 - 1/e)of the steady state response after a step input to the system. A periodof 5 time constants is regarded as the time required for a purefirst-order system to reach 99.3% of the steady state response, at whichpoint the system can generally be regarded as having achieved steadystate. Thus, if the time constant of a first-order system response to astep input can be determined in real time, the time required for thesystem to reach steady state can be reasonably predicted. The timeconstant can be determined in real time if the magnitude of the changein system response to a step input is known. The magnitude of change insystem response to a step input from a positive or negative value tozero can be determined as the difference between the system responsemeasured just before a step input and a known steady state equilibriumresponse to zero input.

During a pipetting operation, beginning at the time the pump 2 stopsactuation, the working pressure decays in an exponential fashion (i.e.,exponential decay 801) as it returns to steady state condition 802. Insome methods disclosed herein, a time constant of this pressure responsein exponential decay is determined in real time in order to predict thetime required to reach steady state condition 802, at which point theaspiration or dispense is considered complete. This is possible becausethe magnitude in change in working pressure can be determined using thereference pressure 803 (P₀), measured before the pipetting operationbegan, as the equilibrium value and by using the working pressuremeasured at the time the pump 2 stops actuation as the initial value ofthe characteristic first-order response. By measuring the time constantof this decaying pressure response, the time at which the responsesettles to steady state equilibrium can be extrapolated. In this way,regardless of the properties of the liquid 61 being aspirated ordispensed, the pressure response provides enough information to predictwhen the pipetting operation will be complete.

In the case of the liquid handling apparatus 1, a variety of factors canaffect the exponential decay of the pressure response during the secondphase of an aspiration or dispense. The main factors include theproperties of the liquid 61 to be aspirated or dispensed, the geometryand design of the pipetting apparatus, and the geometry and design ofthe pipette tip 3. Some factors can have minor effects that vary betweenpipetting operations, such as the volume of liquid in the pipette tip 3at any given time. Environmental factors external to the liquid handlingapparatus 1, such as temperature, ambient pressure, and humidity, canalso influence the pressure response. A unique consideration of thepipetting system, when considered as a first-order system, is residualvacuum of the enclosed air volume 810 when liquid exists in the pipettetip 3 at steady state condition 802. If liquid is in the pipette tip 3at steady state equilibrium, for example after an aspiration operation,a slight negative working pressure (i.e. a vacuum relative to ambientpressure outside the pipette tip 3) exists in the liquid handlingapparatus 1 at steady state, which retains the liquid 61 in the pipettetip 3. The magnitude of this residual vacuum depends on the volume ofliquid in the pipette tip 3, the density of that liquid, and the designof the pipette tip 3, and the volume of air inside the liquid handlingapparatus 1. Thus, the residual vacuum and the magnitude of the finalsteady state response can vary from operation to operation.

Because of the unique considerations of the liquid handling apparatus 1,in one embodiment the time constant (τ) is modified from its generallyaccepted definition to suit the characteristics of a particularpipetting system design. In one example, the time constant is defined asthe period of time required for the pressure response to reach 40% ofthe steady state response. For the purposes of calculating the workingpressure after a period of one time constant, steady state is consideredequal to the ambient reference pressure measured before the operation.In one example, at the time the syringe piston completes its motion forthe desired volume (at pump actuation completion time 807), the workingpressure measurement is recorded as P₁ and the time is recorded as t₁.The working pressure level after a time period of one time constant(P_(τ)) is calculated by:

P_(τ) = P₁-0.4 * (P₁-P₀)

Where

-   P₀ is the reference pressure measured before pump actuation (the    reference pressure 803) and-   P₁ is the pressure measured at the end of pump actuation and    beginning of exponential decay 801 of the working pressure.

The working pressure is continuously monitored as it decays (exponentialdecay 801) until the value of P_(τ) is reached, at which point the timeis recorded as t_(τ) 808. The time constant τ is then calculated as thedifference between t₁ and t_(τ):

τ = t_(τ) − t₁

In one example, the multiple of time constant periods to pass beforesteady state is achieved after a step input must be modified along withthe time constant definition to suit the characteristics of a particularpipetting system design. Continuing the example above, with a timeconstant determined at 40% decay to steady state, steady state can beachieved after a period of 10 time constants, in a system in accordancewith one embodiment. The parameters of the time constant definition andthe multiple of time constants to reach steady state are bound to eachother and can vary based on system design. These parameters can bedetermined empirically.

Because of the numerous mentioned influences on the system, the pressureresponse can vary slightly from operation to operation even for the sameliquid aspirated with the same control parameters. To overcome thisaspect and improve the reliability of the disclosed method, the steadystate prediction can be confirmed by monitoring the rate of change ofthe pressure response over time. In one example, the pressure responserate of change is calculated over a certain time interval. The rate ofchange, s, is calculated as follows by dividing the difference inpressure before and after each time interval by the duration of the timeinterval.

$s\mspace{6mu} = \mspace{6mu}\frac{P_{n} - P_{n - 1}}{t_{s}}$

Where

-   s is the rate of change of the pressure response,-   P is the pressure measured at a particular interval of time,-   n is the number of time intervals that have passed since the    beginning of pressure response rate of change measurement, and-   t_(s) is the rate of change interval of time.

In one example, when the rate of change of the pressure response fallsbelow a threshold, meaning the pressure response is changing very littleover a period of time, the system can be confirmed to have reachedsteady state. Thus, in accordance with one embodiment of the presentlydisclosed method, two conditions must be met to ensure completion of thepipetting operation: the system must wait a period of 10 time constantsafter pump actuation completion time 807, and the system must confirmthe pressure response has settled to steady state condition 802 bywaiting until the rate of change of the pressure response has fallenbelow a threshold. FIG. 3B illustrates this principle, where curve 850in plot 310 shows the slope of the working pressure calculated by thesystem throughout the same 200 uL aspiration of water shown in plot 300of FIG. 3A. Plot 310 of FIG. 3B is synchronized with plot 300 of FIG.3A, and illustrates the features of the slope (rate of change) responsewith relation to key parameters such as the time constant time 808,predicted end time 809, and confirmed end time 802. The confirmed endtime 802 is determined from this slope measurement, when the slope fallsbelow a certain threshold. Additionally, in one example, themeasurements from the pressure sensors 9 are filtered by calculating amoving average of the pressure sensor signal. Preferably, pressuremeasurements from the pressure sensors 9 are made continuously at aconstant data rate. The moving average is determined by calculating theaverage of all previous measurements within a certain window of time ofthe present measurement. Preferably, the output of the filtered pressuresignal is the pressure value considered in the pressure responserate-of-change calculation above.

In one example, the above described parameters (e.g., the time window ofthe moving average filter, the time interval of the pressure responserate of change calculation, and the pressure response rate of changethreshold to confirm steady state) are determined as functions of thetime constant τ. This enables the optimal system response regardless ofthe properties of the liquid 61 being aspirated or dispensed. Forexample, a very viscous liquid will have a relatively long time constantand an associated very long exponential decay to steady state. Near theend of this decay, the pressure response will change very slowly, so therate of change must be measured over a long period of time in order togain sufficient resolution. In comparison, an aqueous solution will havea relatively short time constant and will settle to steady state veryquickly, perhaps faster than the period of time over which the rate ofchange is measured for a viscous liquid. If the optimal parameters ofthe viscous liquid aspiration were used to determine steady state for anaqueous solution aspiration, the operation would not be very efficient.If the optimal parameters of the aqueous solution aspiration, whichinclude a short period of time over which the rate of change ismeasured, were used to determine steady state for a viscous liquid, thesteady state might be mistakenly determined very early resulting in aninaccurate pipetting result.

In one example, the above described parameters are determined as linearfunctions of the time constant τ with empirically determinedcoefficients. The coefficients can be calibrated for a specific systemdesign, and are dependent upon factors including the volume of airbetween the pump 2 and the pipette tip 3, the size and design of thepipette tip 3, and parameters of the pump actuation. The aboveparameters are calculated by:

t_(f) = c₁* τ + c₂

t_(s) = c₃* τ + c₄

s₀ = c₅ * τ + c₆

Where

-   t_(f) is moving average filter time window,-   t_(s) is the pressure response rate of change determination time    interval,-   s₀ is the pressure response rate of change threshold for steady    state, and-   c₁, c₂, c₃, c₄, c₅, and c₆ are empirically determined coefficients    specific to a particular pipetting system design.

In some embodiments, additional control parameters can be derived fromthe time constant in a manner similar to those described above, such asspeed of pipette tip 3 removal from the liquid 61 after the steady stateresponse has been reached. Further, in some embodiments, controlparameters for the dispensation operation can be derived from the timeconstant determined during the associated aspiration operation.

Referring now to FIG. 5 , a flow diagram of an example of a method 500of automatic liquid transfer optimization pipetting is described usingthe presently disclosed the liquid handling apparatus 1, according to asimple configuration. The method 500 can be used to emulate and optimizethe way a human would operate a manual pipette. The method 500 caninclude, but is not limited to, the following steps.

At a step 510, an automatic liquid transfer optimization pipettingapparatus is provided. For example, the liquid handling apparatus 1shown in FIG. 1 and/or FIG. 2 is provided.

At a step 515, using the electronic controller 7 and/or the optimizationalgorithm 8, pump 2 is controlled to limit the working pressure withinsafe limits as a way to ensure accurate error-free pipetting with noprior knowledge of liquid properties.

At a step 520, using the electronic controller 7 and/or the optimizationalgorithm 8, pump 2 is actuated to displace a volume of air required toaspirate or dispense an associated volume of liquid.

At a step 525, using the electronic controller 7 and/or the optimizationalgorithm 8, the pressure response inside the pipette tip 3 afteractuation of the pump 2 is treated as a first-order system response.Namely, a prediction of end time is made based on the time constantvalue, which is determined “on the fly,” and the prediction is confirmedwhen the rate of change of the pressure response falls below a certainthreshold. The parameters for measuring the rate of change and theassociated threshold are determined as a function of the time constantvalue.

Referring now to FIG. 6 , a flow diagram of a method 600 describes aspecific method of automatic liquid transfer optimization pipettingusing a liquid handling apparatus 1 described herein. The method 600 caninclude, but is not limited to, the following steps.

At a step 610, an automatic liquid transfer optimization pipettingapparatus is provided. For example, the liquid handling apparatus 1shown in FIG. 1 and/or FIG. 2 is provided.

At a step 615, a container of liquid to be processed is provided andwherein properties of the liquid are unknown. For example, the container6 is provided that is holding a quantity of the liquid 61, wherein thephysical properties of the liquid 61 are unknown.

At a step 620, the pipette tip of the automatic liquid transferoptimization pipetting apparatus is inserted into the liquid foraspiration or dispensation. For example, under the control of theelectronic controller 7 and/or the optimization algorithm 8, the pipettetip 3 of the liquid handling apparatus 1 is inserted into the liquid 61for aspiration or dispensation.

At a step 625, a reference pressure is determined. For example, usingthe electronic controller 7 and/or the optimization algorithm 8, areference pressure is determined prior to aspiration or dispensation ofthe liquid 61, wherein the reference pressure is equal to the ambientpressure within the conduit 4 and outside the conduit 4 before anyaspiration or dispensation operations.

At a step 630, a pump of the automatic liquid transfer optimizationpipetting apparatus is actuated for aspiration or dispensation of theliquid. For example, under the control of the electronic controller 7and/or the optimization algorithm 8, the pump 2 of the liquid handlingapparatus 1 is actuated for aspiration or dispensation of the liquid 61.More specifically, when the pump 2 is a syringe pump, the piston of thesyringe pump is moved outward (i.e., away from the pipette tip 3) duringan aspiration, and wherein the piston of the syringe pump is movedinward (i.e., toward the pipette tip 3) during a dispensation. Further,in this step, pump actuation is done at a sufficient speed to create asubstantially measureable pressure change within the conduit 4.

At a step 635, the pressure response within a conduit is monitored. Forexample, the electronic controller 7 and/or the optimization algorithm 8are used to monitor readings from the pressure sensors 9 and therebymonitor the pressure response within the conduit 4 of the liquidhandling apparatus 1.

At a step 640, a working pressure of the automatic liquid transferoptimization pipetting apparatus is determined. For example, using theelectronic controller 7 and/or the optimization algorithm 8, a workingpressure of the conduit 4 of the liquid handling apparatus 1 isdetermined.

At a step 645, the working pressure is maintained within predeterminedlimits through the control of a velocity of movement of the pump of theautomatic liquid transfer optimization pipetting apparatus. For example,under the control of the electronic controller 7 and/or the optimizationalgorithm 8, the working pressure in the conduit 4 is maintained withinpredetermined limits through the control of the velocity of movement ofthe pump 2 of the liquid handling apparatus 1.

At a step 650, actuation of the pump is halted after displacement of avolume of air required to aspirate or dispense an associated volume ofliquid. For example, under the control of the electronic controller 7and/or the optimization algorithm 8, actuation of the pump 2 is haltedwhen the pump 2 has displaced a volume of air in the pipette tip 3 thatcorresponds to a desired volume of the liquid 61 that is being aspiratedor dispensed. Thus, when actuation of the pump 2 stops, liquid may stillbe aspirated or dispensed until equilibrium (i.e. steady state) isreached. Further, halting of the pump actuation is done with sufficientdeceleration of the velocity of the pump 2 to act as a step input tocreate a substantially measurable first-order response to the workingpressure.

At a step 655, the steady state pressure response within the conduit ofthe automatic liquid transfer optimization pipetting apparatus isconfirmed. For example, using the electronic controller 7 and/or theoptimization algorithm 8, the steady state pressure response within theconduit 4 of the liquid handling apparatus 1 is confirmed by measuringthe slope of the pressure response over time.

Referring now again to FIG. 5 and FIG. 6 , throughout the steps of themethod 500 and/or the method 600 of automatic liquid transferoptimization pipetting, the liquid transfer process is monitored andautomatically adjusted by the electronic controller 7 and/or theoptimization algorithm 8 to ensure error-free operation and to predictand confirm the time needed to finish the process for maximum accuracy,precision, and efficiency. By utilizing the pressure response rate ofchange to confirm the steady state at the end of the process, the methodis less susceptible to variations caused by environmental conditions,normal differences in fluid properties, and physical variance of systemcomponents.

The presently disclosed liquid handling apparatus 1 and the methods 500,600 can in some embodiments simplify the user experience of automatedpipetting devices and lower the barrier to entry for use of automatedpipetting devices by enabling such devices to pipette any liquid withoutprior knowledge of the liquid properties. For example, as compared withconventional automated liquid handling instruments, the presentlydisclosed liquid handling apparatus 1 and the methods 500, 600 provide ameans to reduce or entirely eliminate (1) the need to specify liquidproperties when programming an automated liquid handler, and (2) theneed to calibrate pipetting parameters for each specific liquid to beprocessed.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a subject” includes aplurality of subjects, unless the context clearly is to the contrary(e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be nonlimiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing amounts, sizes, dimensions,proportions, shapes, formulations, parameters, percentages, quantities,characteristics, and other numerical values used in the specificationand claims, are to be understood as being modified in all instances bythe term “about” even though the term “about” cannot expressly appearwith the value, amount or range. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the followingspecification and attached claims are not and need not be exact, but canbe approximate and/or larger or smaller as desired, reflectingtolerances, conversion factors, rounding off, measurement error and thelike, and other factors known to those of skill in the art depending onthe desired properties sought to be obtained by the presently disclosedsubject matter. For example, the term “about,” when referring to a valuecan be meant to encompass variations of, in some embodiments ± 100%, insome embodiments ± 50%, in some embodiments ± 20%, in some embodiments ±10%, in some embodiments ± 5%, in some embodiments ± 1%, in someembodiments ± 0.5%, and in some embodiments ± 0.1 % from the specifiedamount, as such variations are appropriate to perform the disclosedmethods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or morenumbers or numerical ranges, should be understood to refer to all suchnumbers, including all numbers in a range and modifies that range byextending the boundaries above and below the numerical values set forth.The recitation of numerical ranges by endpoints includes all numbers,e.g., whole integers, including fractions thereof, subsumed within thatrange (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5,as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like)and any range within that range.

Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

1. An apparatus for automatic liquid transfer optimization pipetting,comprising: a pump; a pipette tip in fluid communication with the pump,the pipette tip comprising a conduit with a working air pressurerelative to ambient pressure, the working air pressure having an upperthreshold; a pressure sensor connected to the conduit, the pressuresensor being adapted to measure the working air pressure, the ambientpressure, and changes to the working air pressure caused by aspirationor dispensation of a liquid by the pipette tip; and a controller inelectrical communication with the pump and the pressure sensor, whereinthe controller receives input from the pressure sensor, commands avelocity of the pump, and maintains the working air pressure at or belowthe upper threshold by adjustment of the velocity of the pump duringaspiration or dispensation of a liquid by the pipette tip.
 2. Theapparatus of claim 1, further comprising: a frame; and an actuator fixedto the frame and in electrical communication with the controller, theactuator being operatively connected to the pump and the pipette tip andadapted to control movement of the pump, the pipette tip, or both. 3.The apparatus of claim 1, wherein the pump comprises: a motor; and asyringe operatively connected to the motor.
 4. A method of automaticliquid transfer optimization pipetting, comprising: providing anapparatus according to claim 1; aspirating or dispensing a liquid in theconduit of the pipette tip; adjusting a velocity of the pump to limitthe working pressure in the conduit of the pipette tip to a pressurelevel equal to or less than a maximum working pressure value duringaspiration or dispensing of the liquid; actuating pump to displace avolume of air in the conduit, the volume of air corresponding to adesired volume of the liquid being aspirated or dispensed.
 5. The methodof claim 4, further comprising: providing a container holding theliquid, the liquid having unknown physical properties; and inserting thepipette tip into the liquid for aspiration or dispensation.
 6. Themethod of claim 4, further comprising determining a reference pressurein the conduit, the reference pressure being a pressure in the conduitduring an absence of any liquid in the conduit.
 7. The method of claim6, wherein the reference pressure is equal to an ambient pressure withinthe conduit and outside the conduit.
 8. The method of claim 4, whereinaspirating or dispensing the liquid comprises actuating the pump.
 9. Themethod of claim 4, further comprising monitoring pressure within theconduit with the pressure sensor.
 10. The method of claim 9, furthercomprising the step of filtering the measurements by the pressure sensorby calculating a moving average of the pressure sensor signals.
 11. Themethod of claim 10, wherein the moving average is determined bycalculating the average of all prior measurements within a period oftime.
 12. The method of claim 4, wherein the controller adjusts thevelocity of the pump during actuation to maintain the working pressurein the conduit at a pressure level equal to or less that the maximumworking pressure.
 13. The method of claim 4, further comprisingconfirming a steady state pressure response within the conduit after thepump has been actuated to displace the volume of air in the conduit. 14.The method of claim 13, wherein confirming the steady state pressureresponse comprises measuring the slope of the pressure response overtime.
 15. The method of claim 14, wherein the slope is calculated bydividing the difference in pressure before and after a time interval bythe duration of the time interval, using the formula:$s\, = \,\frac{P_{n}\, - \, P_{n - 1}}{t_{s}}$ wherein s is the slope(time rate of change) of the pressure response, n is the number of timeintervals that have passed since the beginning of slope measurement, Pis the pressure, and t_(s) is the slope interval of time.
 16. The methodof claim 4, wherein the pump comprises a syringe and a piston, andwherein actuating the pump comprises moving the piston outward from thesyringe during an aspiration, and moving the piston into the syringeduring a dispensation.
 17. The method of claim 4, wherein the actuatingstep is done at a sufficient speed to create a substantially measureablepressure change within the conduit.
 18. The method of claim 4, whereinthe working pressure after a period of exponential decay of one timeconstant is calculated as: P_(τ) = P₁ - 0.4 *  (P₁- P₀) wherein P_(τ) isthe working pressure, P₀ is the reference pressure, and P₁ is thepressure at the end of the piston movement and the beginning of theexponential decay.
 19. The method of claim 18, further comprising thestep of continuously monitoring the working pressure as the workingpressure decays until the value of the P_(τ) is reached.
 20. The methodas recited in claim 4, wherein termination of the pump actuation is witha deceleration of the velocity of the pump that acts as a step input tocreate a substantially measurable first-order response to the workingpressure.
 21. The method of claim 4, further comprising the step ofdetermining parameters to adapt the liquid to the pipetting apparatus.22. The method of claim 21, wherein the parameters are parameters arecalculated by: t_(f) = c₁ * τ + c₂ t_(s) = c₃ * τ + c₄ s₀ = c₅ * τ + c₆wherein tf is filtering moving average time window, t_(s) is the slopedetermination time window, s₀ is the slope threshold for steady state,and c₁, c₂, c₃, c₄, c₅, and c₆ are empirically determined coefficientsspecific to a particular pipetting system design.